Nucleotides labelled with an infrared dye and their use in nucleic acid detection

ABSTRACT

Nucleoside-5′-triphosphates and phosphoramidites which carry a residue absorbing in the long wavelength region, preferably a carbocyanine group of the general formula (I), on the base portion or on the phosphorus atom                    
     in which R 1  and R 2  each denote hydrogen or together form a phenyl residue; R 3  denotes hydrogen if linkage with the nucleotide is via the R 4  position or it denotes a —NHCS— group if linkage with the nucleotide is via the R 3  position; both R 4  and R 5 , or R 5 , alone denote an alkylsulfonyl group with n being a number from 3 to 5 or R 4  represents a —NHCS— group with n being a number from 3 to 8, as well as the use of the compounds to label, detect and sequence nucleic acids.

FIELD OF THE INVENTION

The invention concerns nucleoside-5′-triphosphates and phosphoramiditeswhich carry a fluorescent residue absorbing in the long wavelengthrange, preferably a carbocyanine group, on the base portion or on thephosphorus atom, as well as their use for labelling, detecting andsequencing nucleic acids.

BACKGROUND OF THE INVENTION

Nucleic acids are of crucial importance in living nature as carriers ortransferrers of genetic information. Since their discovery by F.Miescher they have therefore stimulated a broad scientific interestwhich has led to the elucidation of their function, structure andmechanism of action. with increasing knowledge of these fundamentalmolecular biological mechanisms it has in recent years become possibleto pursue the new combination of genes. This technology opens forexample new possibilities in medical diagnosis and therapy and in plantbreeding.

An essential tool for understanding these interrelations and solving theproblems was and is the detection of nucleic acids and their sequencesi.e. their primary structure.

The specific detectability of nucleic acids is based on the propertiesof these molecules to interact, i.e. to hybridize, with other nucleicacids by forming base pairs via hydrogen bridges. Nucleic acids (probes)labelled in a suitable manner, i.e. provided with indicator groups, canthus be used to detect complimentary nucleic acids (target).

The determination of the primary structure (sequence), i.e. the sequenceof the heterocyclic bases, of a nucleic acid is carried out by means ofsequencing techniques. This knowledge of the sequence is in turn aprerequisite for a targetted and specific use of nucleic acids inmolecular biological investigations and working techniques. Thesequencing finally also utilizes the principle of specific hybridizationof nucleic acids among each other. As mentioned above, labelled nucleicacid fragments are also used for this.

It is clear from the aforementioned that suitable labelling of nucleicacids is an essential prerequisite for any method of detection.

Above all, radioactive labelling with suitable isotopes such as ³²p or³⁵S is already being used for this at an early stage. The disadvantagesof using radioactive reagents are, however, obvious: such work requiresspecial room installations and permits, as well as a controlled andcomplicated disposal of the radioactive waste. The reagents forradioactive labelling are expensive. A longer storage of such labelledsamples is not possible due to the short half-life of the abovenuclides.

Therefore in recent years there have been attempts to circumvent theseserious disadvantages i.e. to get away from radioactive labelling. Indoing so the high sensitivity of this type of labelling should bepreserved as far as possible. Great advances have in fact been made inthis case [see e.g. Nonradioactive Labeling and Detection ofBiomolecules, C. Kessler (Editor), “Springer Verlag Berlin, Heidelberg”1992].

Haptens (such as biotin or digoxigenin), enzymes (such as alkalinephosphatase or peroxidase) or fluorescent dyes (such as fluorescein orrhodamine) have above all proven to be successful among others asnon-radioactive indicator molecules.

Although labelling with haptens such as e.g. digoxigenin extends intothe sensitivity range of radioactivity, a direct detection ofhapten-labelled nucleic acids analogous to radioactive labelling is notpossible. A subsequent detection reaction is necessary which is, forexample, achieved by means of an antibody reaction. This indirectdetection requires several steps i.e. more time and financial expense.Since proteins are used for the detection reaction, a special treatmentof the solid phase (membranes, microtitre plates) by blocking andwashing steps is necessary in order to reduce unspecific binding.Despite this, the sensitivity of this two-step detection is usuallylimited due to the occurrence of interfering background colourationresulting from unspecific protein binding. The same basically applies todirect enzyme-labelled nucleic acids.

The said disadvantage of the aforementioned indirect detection does notoccur when using fluorescent-labelled nucleic acids. A direct detectionis possible by exciting the fluorescence and can be visualized andmeasured with a suitable device (fluorescence microscope, scanner).However, the autofluorescence of cell and tissue components of thebiological material to be examined such as dyes, lipids, proteins etc.also interferes in this case. Such interferences also occur particularlywhen using solid carrier materials (e.g. nylon membranes) due to theirintrinsic fluorescence and complicate or prevent the detection.

In principle a solution to these problems is to use dyes whoseexcitation and emission is in wavelength ranges above 680 nm i.e. in thenear infrared (NIR) range. The aforementioned interfering influences arenot significant under these circumstances. A further important advantageis that very durable cheap laser diodes can be used for the excitation.

Thus for example the technique of DNA sequencing by photoelectricmeasurement with a laser and a sensor after fluorescent labelling of theDNA fragments is the subject matter of an application U.S. Pat. No.4,729,947. In this method, oligonucleotides labelled with an IR dye areused in a known manner as a primer in the so-called Sanger method whichact in this process as starters for the synthesis of the newcomplementary nucleic acid strand. However, a disadvantage of thismethod is that—depending on the DNA to be sequenced—specific labelledprimers have in each case to be newly synthesized again and again i.e.numerous such labelled primers. This synthesis of labelled oligomericprimers is expensive and time-consuming since the unlabelledoligonucleotide has to be synthesized at first and subsequently thesignal (reporter) group is chemically attached in a second reaction.

The object of the present invention is therefore to produce compoundswhich enable a universal, simple and specific labelling of nucleicacids.

It is now known that nucleic acids can be newly synthesized andconcomitantly labelled by the incorporation of appropriately labellednucleoside triphosphates using polymerases. In the field ofdeoxyribonucleic acids (DNA) this is achieved by DNA polymerases usingthe methods of nick translation [Rigby, P. W. et al. (1977) J. Mol.Biol. 113, 237] and of random primed labelling [Feinberg, A. P. &Vogelstein, B. (1984) Anal. Biochem. 137, 266] by incorporatingdeoxynucleotides and in the case of ribonucleic acids by RNA polymerasesand ribonucleotides along the lines of a transcription. A further methodof labelling nucleic acids is by means of a so-called 3′ tailingreaction with the aid of terminal transferase and ribo ordeoxyribonucleoside triphosphates.

However, nucleoside triphosphates provided with indicator molecules suchas fluorescein or digoxigenin (MW 332 or 390) are—in contrast to theirnatural substrates—accepted relatively poorly as substrates bypolymerases and incorporated relatively poorly into the newlysynthesized nucleic acid (Hoeltke, H.-J. et al. (1990) Biol. Chem.Hoppe-Seyler 371, 929).

It is therefore not to be expected that indicator molecules with evenconsiderably higher molecular weights (800-1000) would be accepted bypolymerases as substrates and incorporated into nucleic acids. It iseven less likely that these molecules with their given spatiallydemanding structure would be converted by polymerases due to strongsteric hindrance.

Surprisingly it has been found that nucleoside triphosphates labelledwith infrared dyes are accepted as substrates by polymerases such as T7DNA polymerase and are incorporated into nucleic acids. The compoundsaccording to the invention are therefore novel.

A further object of the invention is to provide a method of using theaforementioned labelled nucleotides according to the invention whichenable nucleic acids labelled thus to be detected directly on solidcarriers such as e.g. nylon membranes or in solution such as e.g. inmicrotitre plates.

As already described above, a disadvantage of labelling nucleic acidswith fluorophores such as fluorescein or tetramethylrhodamine is thatthe intrinsic fluorescence of the carrier material interferes with themeasurement of this fluorescence.

If, however, the nucleoside triphosphates labelled with IR dye accordingto the invention are used to label nucleic acids, these interferencesare no longer significant because the measurement wavelength is locatedin the near infrared range.

The advantages of a nucleic acid detection by in situ hybridization areknown. In this method the labelled samples or probes are either detecteddirectly under a fluorescence microscope or, in the case of haptenlabelling, are detected in an immunological reaction (ELISA) by means ofa further process step. This visualization is usually achieved byimmobilizing the samples for example on a nylon membrane or in a liquidhomogeneous phase in microtitre plates. This additional step is verytime-consuming and costly. It is thus desirable to omit this step.

SUMMARY OF THE INVENTION

In the instant invention, the possibility of directly exciting theIR-fluorescent-labelled nucleic acids by suitable laser diodes and theaforementioned insensitivity of the detection towards autofluorescenceof the carrier material enables simple and cost-effective equipment tobe used. The immunological detection reaction can be omitted. TheIR-labelled nucleic acid is simply detected by optical means through alaser/detector combination with the aid of a suitable scanner ormicrotitre plate reader.

The use of the nucleoside 5′-triphosphates labelled with infraredfluorophores as polymerase substrates enables direct enzymaticincorporation into nucleic acids and detection of the nucleic acidslabelled in this manner for sequencing, and it also allows an in situhybridization which is also part of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The nucleoside 5′-triphosphates of the invention are of the generalformula

They are produced by starting with unmodified nucleosides in awell-known manner, these i.e. uridine, thymidine and cytidine in thecase of pyrimidine nucleosides and the purine nucleosides adenosine andguanosine as well as the corresponding 7-deaza-purine and7-deaza-8-aza-purine nucleosides, are chemically modified in a suitablemanner at C-5 or C-6 (pyrimidine), at C-8 (purine), at C-8(3-deaza-purine) at C-7 or C-8 (7-deaza-purine) and finally5′-phosphorylated.

It is expedient that the modified group is composed of a spacer ofsuitable length and a terminal primary or secondary amino group whichcan be substituted by suitable activated fluorescent dyes (e.g. in theform of isothiocyanates or N-hydroxysuccinimide esters).

Such fluorescent dyes are used in an activated form, i.e. in a formwhich reacts well with for example amino groups, preferably asisothiocyanates. After the reaction is completed the fluorophores arecovalently bound via NHCS groups to the modified group of thenucleotide.

The phosphorylation of the nucleosides modified in this way is carriedout according to methods known in the literature [e.g. Yoshikawa, M. etal. (1967) Tetrah. Lett. 50, 5065] by reaction with phosphoryltrichloride to form the monophosphate and subsequent reaction withpyrophosphoric acid to form the desired 5′-triphosphate. As analternative a direct modification of the preformed nucleoside5′-triphosphates is also possible.

The fluorophores are compounds which absorb in the near infrared rangei.e. between 600 and 800 nm. Those of 630 nm to 780 nm are preferredsuch as e.g. carbocyanines.

As already mentioned above, in addition to the method described above ofincorporating labelled nucleoside triphosphates using polymerases, afurther method is common which is based on the use of labelledoligonucleotides, so-called primers. As already mentioned, theconventional synthesis of these molecules in a multistep reaction isvery time-consuming. In this process the signal group must be attachedat the 5′-end of the oligomer in an additional step after theoligonucleotide synthesis is completed. Since the actual oligonucleotidesynthesis is carried out in automatic synthesizers, it is extremelydesirable to also be able to carry out the step of attaching the signalgroup already in the synthesizer. Since the oligonucleotide synthesis iscomposed of a stepwise attachment of monomeric building blocks,so-called nucleoside phosphoramidites, it is a further object of theinvention to develop a fluorophore phosphoramidite which enables thedirect incorporation of the signal group as the last step in automaticoligonucleotide synthesis. Such a NIR-dye-phosphoramidite is hithertounknown and therefore novel.

The invention is elucidated in more detail by the following examples.

EXAMPLE 1 5-(3-Aminoallyl)-2′-deoxy-uridine-5′-triphosphate

This derivative was synthesized as described by Langer et al in Proc.Natl. Acad. Sci. USA (1981) 78, 6635.

EXAMPLE 2Anhydro-11-phenoxy-10,12-propylene-3,3,3′,3′-tetramethyl-4,5-benzindo-indotricarbocyanin-1-(4-sulfobutyl)-1′-(3-aminopropyl)-thiono-[5-(3-aminoallyl)-2′-deoxyuridine-5′triphosphate]

A solution of 50 mganhydro-11-phenoxy-10,12-propylene-3,3,3′,3′-tetramethyl-4,5-benzindo-1-(4-sulfobutyl)-1′-(3-isothiocyanopropyl)-indotricarbocyanineNa salt (60 μmol) in 1 ml dimethylformamide is added to a solution of 33mg 5-aminoallyl-dUTP-Li₄ (60 μmol) in 2 ml 0.1 M Na-borate buffer, pH8.5 and the reaction mixture is allowed to stand for about 15 hours atroom temperature while protected from light. Afterwards the majorportion of the starting materials have reacted according to paperelectrophoresis (0.05 M Na-citrate buffer, pH 5). In order to isolatethe desired substance, the reaction mixture is diluted with about 50 mlwater and the deep-green coloured solution is applied to an ion exchangecolumn containing DEAE Sephadex A-25 in the chloride form. The productis eluted from the column with a linear gradient of water to 1 M LiCl,and the product fractions are concentrated in a vacuum and desalted bymeans of reversed phase chromatography on RP 18 material. Afterlyophilization, 6 μmol (10%) of the desired triphosphate is obtained.

Spectral data: Emission_(max) 786 nm, 720 nm (shoulder), 238 nm.

EXAMPLE 3Anhydro-10,12-propylene-3,3,3′,3′-tetramethyl-1,1′-bis(3-sulfobutyl)-indotricarbocyanin-11-(4-amino)-phenoxy-thiono-[8-(5-aminopentylamino)-2′-deoxy-adenosine-5′-triphosphate](“IRD-dATP”)

The derivative is prepared according to the process set forth in example2 from 38 mg 8-aminopentylamino-dATP (60 μmol) and 50 mganhydro-10,12-propylene-3,3,3′,3′-tetramethyl-1,1′-bis(3-sulfobutyl)-11-(4-isothiocyano)-phenoxy-indotricarbocyanineNa salt (60 μmol). 3 μmol of the compound was obtained.

Spectral data: Emission_(max) 770 nm, 697 nm (shoulder), 279 nm.

EXAMPLE 4 Use of IRD-dATP as a Substrate for T7-DNA Polymerase

3 μg template DNA is incubated for 15 minutes at 37° C. in a mixture of2 μl reaction buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl₂, 250 mMNaCl), 1 pM M13/pUC primer and 7 μl H₂O.

1 μl DTT (100 mM), 2 μl labelling mixture (10 μM IRD 40-dATP, 1 μM eachof dCTP, dGTP and dTTP), 1 μl H₂O and 2 μl T7-DNA polymerase (2.5 U/μl)are added for the labelling reaction and it is incubated for 10 minutesat room temperature.

For use in DNA sequencing, a termination reaction is subsequentlycarried out by addition of the termination mixture (ddATP, ddGTP, ddCTP,ddTTP).

EXAMPLE 5Anhydro-11-phenoxy-10,12-Propylene-3,3,3′,3′-tetra-methyl-4,5-benzindo-indotricarbocyanine-1-(4-sulfo-butyl)-1′-(3-aminopropyl)-thiono-[5-(3-aminoallyl)-uridine-5′-triphosphate

The compound was synthesized analogously to example 2 from5-aminoallyl-UTP (prepared according to example 1) and the correspondingisothiocyanate.

The spectral data correspond to the 2′-deoxy compound of example 2.

EXAMPLE 6Anhydro-10,12-propylene-3,3,3′,3′-tetramethyl-1,1′-bis(3-sulfobutyl)-indo-tricarbocyanin-11-(4-amino)phenoxy-thiono-[5-(3-aminoallyl)-2′,3′-dideoxy-uridine-5′-triphosphate](“IRD-ddUTP”)

Step 1: 2′,3′-Dideoxy-uridine-5′-triphosphate

The derivative was synthesized via the unstable diazonium derivativestarting with the commercially available2′,3′-dideoxy-cytidine-5′-triphosphate (Boehringer Mannheim) bydeamination with NaNO₂/acetic acid.

Step 2: 5-(3-Aminoallyl)-2′,3′-dideoxy-uridine-5′-triphosphate

The compound was prepared analogously to example 1 according to Langeret al supra via the 5-mercury derivative of 2′,3′-dideoxy-UTP.

Step 3: “IRD-ddUTP”

The dideoxy derivative was obtained according to example 2 by reactingthe 5-aminoallyl-ddUTP with the corresponding isothiocyanate.

The spectral data correspond to those of the 2′-deoxy compound ofexample 3.

EXAMPLE 7Anhydro-10,12-propylene-3,3,3′,3′-tetramethyl-1,1′-bis(3-sulfopropyl)-indo-tricarbocyanin-11-[(4-ethoxy)phenoxy-O-(2-cyanoethyl)-N,N-diisopropl-phosphoramidite

In a 50 ml round bottom flask 425 mganhydro-11-(4-hydroxyethyl)phenoxy-10,12-propylene-3,3,3′,3′-tetramethyl-1,1′-bis(3-sulfopropyl)-indotricarbocyanine-hydroxidein the form of its Na salt (0.5 mmol) is dissolved in 5 ml dryacetonitrile and 0.275 ml ethyldiisopropylamine (1.6 mmol) is added.Subsequently 0.125 mlchloro-β-cyanoethoxy-N,N-diisopropylamino-phosphane are added dropwisewithin about 3 minutes under nitrogen and while stirring. It is stirredfor a further 30 minutes at room temperature, about 10 ml aqueous 5%NaHCO₃ solution is then added and it is subsequently extracted twicewith, about 10 ml dichloromethane each time. The pooled organic phasesare dried over sodium sulfate, the solvent is removed by distillationand the residue is chromatographed on silica gel using the mobilesolvent dichloromethane/ethyl acetate/triethyl-amine 45:45:10.

The yield is 480 mg=88.7% of theory.

TLC (silica gel, mobile solvent as above) R_(f)=0.4 ³¹p-NMR (d₆DMSO):149 and 153 ppm (2 diastereomers).

What is claimed is:
 1. A nucleoside-5′-triphosphate of the generalformula

wherein B is a heterocyclic base selected from the group consisting ofadenine, guanine, hypoxanthine, 7-deaza-adenine, 7-deaza-guanine,7-deaza-hypoxanthine, 7-deaza-8-aza-adenine, 7-deaza-8-aza-guanine,7-deaza-8-aza-hypoxanthine, thymine, cytosine and uracil; x is a linkinggroup wherein n=4-20 atoms; Sig is a fluorescent molecule with anexcitation wavelength of 650-800 nm; and R¹ and R² each represent H orOH.
 2. Method for detecting a target nucleic acid molecule, comprising:(a) contacting said target nucleic acid molecule with a complementarynucleic acid probe under conditions favoring hybridization between saidtarget nucleic acid sequence and said complementary probe, wherein saidcomplimentary nucleic acid probe is characterized as a nucleic acidsequence having incorporated therein at least one labelled nucleoside5′-triphosphate according to claim 1, and (b) detecting said labelledcomplimentary nucleic acid probe as a determinant of said target nucelicacid molecule.
 3. The method of claim 2, wherein said labelled nucleicacids are detected by hybridization.
 4. The method of claim 3, whereinsaid hybridization is carried out on membranes or in solution.
 5. Themethod of claim 2, wherein the labelled nucleic acids are detected usinglaser diodes and detectors.
 6. The method of claim 2, which furthercomprises the step of sequencing said labelled nucleic acids.
 7. Thecompound of claim 1, wherein: x is a linking group wherein n=10-15 atomsand Sig is a carbocyanine of the general formula:

 wherein R¹ and R²=H or together represent a phenyl residue; R³=H iflinkage with the nucleotide is via the R⁴ position, or R³=—NHCS— iflinkage is via the R³ position; and R⁴ and R⁵ each denote alkylsulfonylwherein n=3-5; or R⁴—NHCS— wherein n=3-8 and R⁵=alkylsulfonyl whereinn=3-5 if linkage is via the R⁴ position.
 8. A method of detectingnucleic acids comprising labelling nucleic acids with the compound ofclaim 7, and detecting said labelled nucleic acids.
 9. The method ofclaim 8, wherein said labelled nucleic acids are detected byhybridization.
 10. The method of claim 9, wherein said hybridization iscarried out on membranes or in solution.
 11. The method of claim 8,wherein the labelled nucleic acids are detected using laser diodes anddetectors.
 12. The method of claim 8, which further comprises the stepof sequencing said labelled nucleic acids.
 13. An oligonucleoide whichcomprises the nucleoside molecule of claim
 1. 14. The nucleoside5′-triphosphate of claim 1, wherein said triphosphate is selected fromthe group consisting ofanhydro-11-phenoxy-10,12-propylene-3,3,3′,3′-tetramethyl-4,5-benzindo-indotricarbocyanin-1-(4-sulfobutyl)-1′-(3-aminopropyl)-thiono-[5-(3-aminoallyl)-2′-deoxyuridine-5′-triphosphate]4anhydro-10,12-propylene-3,3,3′,3′-tetramethyl-1,1′-bis(3-sulfobutyl)-indotricarbocyanin-11-(4-amino)-phenoxy-thione-[8-(5-aminoapentylamino)-2′-deoxyadenosine-5′-triphosphate];anhydro-11-phenoxy-10,12-propylene-3,3,3′,3′-tetramethyl-4,5-benzindo-indotricarbocyanine-1-(4-sulfobutyl)-1′-(3-aminopropyl)-thiono-[5-(3-aminoallyl)-uridine-5′-triphosphate];anhydro-10,12-propylene-3,3,3′,3′-tetramethyl-11′-bis(3-sulfobutyl)-indo-tricarbocyanin-11-(4-amino)-phenoxy-thiono-[5-(3-aminoaallyl)-2′,3′-dideoxyuridine-5′-triphosphate];andanhydro-10,12-propylene-3,3,3′,3′-tetramethyl-1,1′-bis(3-sulfopropyl)-indo-tricarbocyanin-11-(4-ethoxy)phenoxy-o-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite.